Method and apparatus for tissue transfer

ABSTRACT

A handheld tool is disclosed which may be used to transfer a plurality of plant tissue explants from a first container to a second container. The handheld tool may include a disposable tip member which couples the plurality of plant tissue explants through use of negative pressure. An automated system which transfers a plurality of plant tissue explants from a first container to a second container is also disclosed. The automated system may include a first presentment system which moves the first container to a region, a second presentment system which moves the second container to the region, and a robot system that transfers the plurality of plant tissue explants from the first container to the second container.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/163,625, filed Mar. 26, 2009, titled “METHOD ANDAPPARATUS FOR TISSUE TRANSFER”, the disclosure of which is expresslyincorporated by reference herein.

FIELD

The present invention relates to methods and apparatus for handlingplant tissue cultures and in particular to methods and apparatus forhandling the transfer of plant tissue culture explants.

BACKGROUND

Many different types of plant tissues are used in a research laboratorysetting. In one example, plant tissue is grown outside of an intactplant as plant tissue cultures. This plant tissue is grown, maintainedor otherwise cultured in an aseptic environment having a nutrient mediumsupporting the plant tissue. The plant tissue is often referred to inthe art as plant tissue explants. One example of a plant tissue explantwould be canola hypocotyl segments cut from germinated seeds.

The claimed methods and apparatus are particularly useful formanipulating plant tissue explants in preparation for recombinant DNAprocedures and for handling explants and callus resulting fromrecombinant DNA techniques. These techniques are used to introduce noveltransgenic events into plant cells to produce fertile plants bearinguseful phenotypes such as herbicide tolerance and insect resistance.

Typically these plant tissue explants are matured in containers havingsolid or semi-solid nutrient medium and observed by trainedprofessionals to determine the usefulness of a given plant tissueexplant. By way of example, a plant tissue explant may be observed orassayed to identify those plant tissue explants that have advantageouscharacteristics or traits introduced by recombinant DNA technology.

Often several plant tissue explants are arranged in a container of solidor semi-solid nutrient substrate supporting the tissue explants. Thesubstrate provides nutrients for the cells within the tissue and isgenerally comprised of a mixture of inorganic salts, vitamins, a carbonsource such as sucrose, and plant growth regulators or hormones. Theplant tissue explants change over time and are observed periodically bya trained professional. Several times during the observation period, thetissue explants must be transferred to a different substrate in order toassure adequate nutrient provision to the tissue explants.

In one example, maize embryos are stored in containers in a room havinga temperature of about 28° C., about 30% humidity, and generally darkfor about a one month period of time. During that one month period oftime, the maize tissue pieces must be transferred to differentcontainers with a different substrate to provide adequate nutrients tothe tissue pieces. In an example, the maize tissue pieces aretransferred once a week. In one example, about 50 individual maizetissue explants are provided in each of 100 containers.

In another example, canola tissue pieces are stored in containers in aroom having a temperature of about 22-23° C. and generally dark forabout 8 hours a day and light for 16 hours a day over about five to sixweeks. During that five to six week period of time, the canola tissuepieces must be transferred to different containers with a differentsubstrate to provide adequate nutrients to the tissue pieces. In anexample, the canola tissue pieces are transferred every week or twoweeks. In one example, about 20 to about 50 individual canola tissuepieces are provided in each of 40 containers. In one example, about 3 toabout 5 mm canola segments are cut from germinated seeds and placed intocontainers each having the nutrient medium. In one experiment from about1000 to about 2000 canola segments are provided (about 20 to about 100containers) are provided. These 1000 to 2000 segments need to betransferred every week or two. Generally up until the fifth transfer,all of the 1000 to 2000 segments are to be transferred.

In a further example, cotton tissue explants are stored in containers ina room having a temperature of about 28° C. and generally dark for about8 hours a day and in indirect lighting for 16 hours a day over aboutthree months. During that three month period of time, the cotton tissueexplants must be transferred to different containers with a differentsubstrate to provide adequate nutrients to the tissue explants. In anexample, the cotton tissue explants are transferred every four weeks. Inone example, about 5 individual cotton tissue explants are provided ineach of 300 containers.

Traditionally, plant tissue explants of a given crop have beentransferred manually by a trained professional from a first container toa second container with forceps in an aseptic environment. The trainedprofessional would place an individual tissue piece between the tongs ofthe forceps, squeeze the tongs together, pick up the individual tissueexplant, and release the pressure on the tongs of the forceps when thetissue piece is located correctly relative to the second container. Aprofessional may transfer up to about 3,000 tissue explants this wayduring a given eight hour work day. This process results in fatigue forthe trained professional over time and potentially an increase in therisk of injury to the trained professional.

In addition, the transfer of tissue explants from the first container tothe second container is performed in an aseptic environment.Traditionally, the transfer is performed in a laminar flow hood, such asthe EDGEGUARD® brand laminar flow hood available from The Baker Companylocated in Sandford, Me. In addition, the forceps used for transferringthe tissue explants must be sterilized between container transfers of agiven set of tissue explants. This prevents cross contamination from onecontainer transfer run (a first set of tissue explants from a firstcontainer to a second container) and a subsequent container transfer run(a second set of tissue explants from a third container to a fourthcontainer). One method used to sterilize the forceps is to submerge thetip of the forceps in EtOH and pass the tongs of the forceps through anopen flame. This creates a risk that the ethanol might ignite resultingin a contamination of the environment. Further, the hood in which thetransfer was being performed may need to be serviced, such as thereplacement of the HEPA filters in the hood. This takes the transferstation out of operation for a period of time.

SUMMARY

In an exemplary embodiment of the present disclosure, apparatus areprovided which reduce the ergonomic injury risk and may increaseproductivity and capacity. Further, the apparatus may reduce the risk ofcontamination.

In another exemplary embodiment of the present disclosure, a method oftransferring plant tissue explants from a first container having a firstnutrient medium substrate adapted to provide nutrients to the planttissue explants to a second container having a second nutrient mediumsubstrate adapted to provide nutrients to the plant tissue explants isprovided. The method comprising the steps of: (a) coupling a first planttissue explant to a handheld tool, the handheld tool coupling the firstplant tissue explant through a negative pressure present in an interiorof the handheld tool; (b) generally separating the first plant tissueexplant from the first nutrient medium substrate adapted to providenutrients to the first plant tissue explant and from the firstcontainer; (c) locating the first plant tissue explant relative to thesecond nutrient medium substrate adapted to provide nutrients to thefirst plant tissue explant of the second container; and (d) uncouplingthe first plant tissue explant from the handheld tool. In an examplethereof, the handheld tool includes a fluid conduit having a first endand a second end. The second end of the fluid conduit being in fluidcommunication with the first end of the fluid conduit and the fluidconduit having at least one vent passage positioned between the firstend of the fluid conduit and the second end of the fluid conduit. Thevent passage being in fluid communication with the first end of thefluid conduit and the second end of the fluid conduit and to thesurrounding environment. The vent passage being blocked from fluidcommunication with the surrounding environment during steps (a) through(c) with a stop device and the vent passage being in fluid communicationwith the surrounding environment during step (d). In a variationthereof, the stop device is one of an operator's finger and a devicesupported by the operator's finger. In another variation, the fluidconduit is an assembly and includes at least a first portion and asecond portion, both the first portion and the second portion having arespective interior in fluid communication with the first end of thehandheld tool and the second end of the handheld tool when the firstportion and the second portion are assembled. In addition, the methodfurther comprises the steps of: (e) providing a plurality of firstportions of the handheld tool; (f) assembling a first one of theplurality of first portions of the handheld tool to the second portionof the handheld tool; (g) performing steps (a) through (d) for all ofthe plant tissue explants in the first container selected for transferto the second container; (h) separating the first one of the pluralityof first portions of the handheld tool from the second portion of thehandheld tool; (i) obtaining a third container including anotherplurality of plant tissue explants for transfer to a fourth container;(j) assembling a second one of the plurality of first portions of thehandheld tool to the second portion of the handheld tool; and (k)performing steps (a) through (d) for all of the plant tissue explants inthe third container selected for transfer to the fourth container. In arefinement thereof, the plurality of first portions are a plurality ofpipette tips. In a further refinement thereof, the plurality of pipettetips are stored generally vertical in a pipette tip box with a tipportion lower than a coupling portion and the step of assembling a firstone of the plurality of first portions of the handheld tool to thesecond portion of the handheld tool includes the step inserting aportion of the second portion of the handheld tool into the couplingportion of a first pipette tip of the plurality of pipette tips whilethe first pipette tip is stored in the pipette tip box. In anotherexample, the steps (a) through (d) are performed in an asepticenvironment. In a variation thereof, steps (a) through (d) are performedin a laminar flow hood. In yet another example, the step of uncouplingthe first plant tissue explant from the handheld tool includes the stepof reducing the negative pressure in the handheld tool by opening a ventpassage of the handheld tool.

In yet another exemplary embodiment of the present disclosure, a systemcoupled to a negative pressure source for use transferring plant tissueexplants from a first container having a first nutrient medium substrateadapted to provide nutrients to the tissue explants to a secondcontainer having a second nutrient medium substrate adapted to providenutrients to the plant tissue explants is provided. The systemcomprising a flexible fluid conduit having a first end adapted to becoupled to the negative pressure source and a second end which is influid communication with the first end through an interior of theflexible fluid conduit; a handheld, pen shaped instrument coupled to thesecond end of the flexible conduit, the handheld pen shaped instrumenthaving an interior in fluid communication with the interior of theflexible fluid conduit; and a tip member removably coupled to thehandheld pen shaped instrument. The tip member having an interior influid communication with the interior of the handheld pen shapedinstrument and a first end having an opening in fluid communication withthe interior of the tip member. The opening of the first end of the tipmember being smaller than the plant tissue explants to prevent the planttissue explants from entering the interior of the tip member when anegative pressure is present in the interior of the flexible fluidconduit, the handheld, pen-shaped instrument, and the tip member. In anexample thereof, the handheld pen shaped instrument includes at leastone vent passage along an exterior of the handheld pen shaped instrumentand in fluid communication with the surrounding environment and theinterior the handheld pen shaped instrument. When the vent passage isnot in fluid communication with the surrounding environment, thenegative pressure in the tip member is increased compared to when thevent passage is in fluid communication with the surrounding environment.In another example, the tip member is a pipette tip. In yet anotherexample, the system further comprises a filter positioned between thefirst end of the flexible fluid conduit and the first end of the tipmember. In a refinement thereof, the flexible fluid conduit is dividedinto a first portion and a second portion. The filter being housed in ahousing which is interposed between the first portion and the secondportion.

In still another exemplary embodiment of the present disclosure, asystem coupled to a negative pressure source for use transferring planttissue explants from a first container having a first nutrient mediumsubstrate adapted to provide nutrients to the tissue explants to asecond container having a second nutrient medium substrate adapted toprovide nutrients to the plant tissue explants is provided. The systemcomprising a flexible fluid conduit having a first end adapted to becoupled to the negative pressure source and a second end which is influid communication with the first end through an interior of theflexible fluid conduit; and a tip member removably coupled to theflexible fluid conduit. The tip member having an interior in fluidcommunication with the interior of the flexible fluid conduit and afirst end having an opening in fluid communication with the interior ofthe tip member. The opening of the first end of the tip member beingsmaller than the plant tissue explants to prevent the plant tissueexplants from entering the interior of the tip member when a negativepressure is present in the interior of the flexible fluid conduit andthe tip member. The tip member further includes at least one ventpassage along an exterior of the tip member and in fluid communicationwith the surrounding environment and the interior the tip member. Whenthe vent passage is not in fluid communication with the surroundingenvironment, the negative pressure in the tip member is increasedcompared to when the vent passage is in fluid communication with thesurrounding environment. In an example thereof, the tip member is apipette tip. In another example thereof, the system further comprises afilter positioned between the first end of the flexible fluid conduitand the first end of the tip member. In a variation thereof, theflexible fluid conduit is divided into a first portion and a secondportion. The filter being housed in a housing which is interposedbetween the first portion and the second portion.

In yet still another exemplary embodiment of the present disclosure, asystem coupled to a negative pressure source for use transferring aplurality of plant tissue explants from a first container having a firstnutrient medium substrate adapted to provide nutrients to the tissueexplants to a second container having a second nutrient medium substrateadapted to provide nutrients to the plant tissue explants is provided.The system comprising a robot system supporting a fluid conduit havingan interior which is in fluid communication with the negative pressuresource; at least one camera positioned to monitor a region including thefirst container and the second container; and a controller operativelycoupled to the at least one camera and the robot system. The controllerbased on input from the at least one camera (a) moves the robot systemto couple a first tissue explant of the plurality of plant tissueexplants in the first container to the fluid conduit through negativepressure in the interior of the fluid conduit, (b) moves the robotsystem such that the first tissue explant is proximate the secondnutrient medium of the second container; and (c) uncouples the firsttissue explant from the fluid conduit such that the first tissue explantis left in the second container. In an example thereof, the controllerrepeats steps (a) through (c) for each of the plurality of plant tissueexplants in the first container. In a variation thereof, each of theplurality of plant tissue explants are placed in the second container toform a predefined pattern. In another example, the system furthercomprises an identification system operatively coupled to thecontroller. The identification system including a reader whichdetermines an identification of the first container and a marker whichprovides identifying indicia on the second container. In yet anotherexample, the system further comprises a first presentment system whichplaces the first container in the region and a second presentment systemwhich places the second container in the region. In a variation thereof,the first presentment system removes the first container from a firstqueue of containers for transfer and places the first container in aqueue of waste containers subsequent to a completion of the transfer ofplant tissue from the first container to the second container. In arefinement thereof, the first presentment system is operatively coupledto the controller. The controller providing instructions to the firstpresentment system regarding the movement of the first container. In afurther refinement thereof, the first presentment system includes aturntable which transports the first container from the first queue ofcontainers for transfer to the region and from the region to the queueof waste containers. In yet another refinement, the second presentmentsystem removes the second container from a second queue of containersfor transfer and places the second container in a third queue ofcontainers subsequent to a completion of the transfer of plant tissuefrom the first container to the second container. In a furtherrefinement, the second presentment system is operatively coupled to thecontroller. The controller providing instructions to the secondpresentment system regarding the movement of the second container. Instill a further refinement, the second presentment system includes aturntable which transports the second container from the second queue ofcontainers for transfer to the region and from the region to the thirdqueue of containers. In yet another example, the fluid conduit includesa removable tip member which interfaces with the plurality of tissueportions. In a variation thereof, the controller prior to steps (a)through (c) selects the removable tip member from a queue of removabletip members and subsequent to completion of steps (a) through (c)discards the removable tip member and selects a second removable tipmember from the queue of removable tip members.

In yet still another exemplary embodiment of the present disclosure, amethod of transferring plant tissue explants is provided. The methodcomprising the steps of: (a) monitoring a region with at least onecamera; (b) providing a first container with a plurality of plant tissueexplants supported on a first nutrient substrate for transfer in themonitored region; (c) providing a second container in the monitoredregion for receiving the plurality of plant tissue explants andsupporting the plurality of plant tissue explants on a second nutrientsubstrate; and (d) automatically transferring the plant tissue explantsfrom the first container to the second container through negativepressure applied by a robot system. In an example thereof, the robotsystem includes a fluid conduit in fluid communication with a source ofnegative pressure and the step of automatically transferring the planttissue explants from the first container to the second container throughnegative pressure applied by a robot system includes the steps of:selecting a tip member from a queue of tip members; coupling the tipmember to the robot system such that a first end of the tip member is influid communication with the fluid conduit; for each plant tissueexplant in the first container: locating the respective plant tissueexplant in the first container; positioning the first end of the tipmember proximate the respective plant tissue explant; coupling therespective plant tissue explant to the first end of the tip member dueto the first end of the tip member being in fluid communication with thesource of negative pressure through a fluid conduit of the tip member;moving the tip member and the respective plant tissue explant to apredefined location relative to the second container; and uncoupling therespective plant tissue explant from the tip member by changing thepressure in the fluid conduit of the tip member; and uncoupling the tipmember from the robot system and discarding the tip member. In anexample thereof, the step of providing a first container with aplurality of plant tissue explants supported on a first nutrientsubstrate for transfer in the monitored region includes the steps of:identifying the first container; selecting the first container from afirst queue of containers; removing a lid of the first container; andmoving the first container to the region being monitored. In anotherexample thereof, the step of providing a second container in themonitored region for receiving the plurality of plant tissue explantsand supporting the plurality of plant tissue explants on a secondnutrient substrate includes the steps of: selecting the second containerfrom a second queue of containers; identifying the second container;removing a lid of the second container; and moving the second containerto the region being monitored.

The above mentioned and other features of the invention, and the mannerof attaining them, will become more apparent and the invention itselfwill be better understood by reference to the following description ofembodiments of the invention taken in conjunction with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a handheld tool for use in transferring plant tissueexplants in an aseptic environment;

FIG. 2 illustrates a exploded view of the handheld tool of FIG. 1;

FIG. 3 illustrates a top view of a tip member of the handheld tool ofFIG. 1;

FIG. 4 illustrates a sectional view of the tip member of FIG. 3 alonglines 4-4 and illustrating air flow through an interior of the tipmember;

FIG. 5 illustrates an operator using the handheld tool of FIG. 1 toselect a first tissue explant in a first container for transfer to asecond container;

FIG. 6 illustrates the sectional view of FIG. 4 with a stop deviceblocking a vent passage of the tip member such that the first tissueexplant is coupled to an end of the tip member;

FIG. 7 illustrates the operator using their finger as the stop devicesuch that the first tissue explant is coupled to the end of the tipmember and is being transferred to the second container;

FIG. 8 illustrates the sectional view of FIG. 4 with a stop devicespaced apart from the vent passage of the tip member such that the firsttissue explant is uncoupled from an end of the tip member;

FIG. 9 illustrates the operator having the first tissue explantpositioned over the second container;

FIG. 10 illustrates a portion of another handheld tool for use intransferring plant tissue explants in an aseptic environment, thehandheld tool including a pen-shaped instrument having a vent passageand a tip member which is coupled to the pen-shaped instrument;

FIG. 11 illustrates the tip member and pen-shaped instrument of thehandheld tool of FIG. 10;

FIG. 12 illustrates the handheld tool of FIG. 10 being used by anoperator;

FIG. 13 illustrates a representative view of an automatic transfersystem for transferring plant tissue explants from a first container toa second container;

FIG. 14 illustrates a representative method of using an automatictransfer system for transferring plant tissue explants from a firstcontainer to a second container;

FIG. 15 illustrates a representative method of providing a firstcontainer in a monitored region, the first container including aplurality of plant tissue explants for transfer to a second container.

FIG. 16 illustrates a representative method of providing a secondcontainer in the monitored region, the second container receiving theplurality of plant tissue explants transferred from the first container;and

FIG. 17 illustrates a representative method of automaticallytransferring the plant tissue explants from the first container to thesecond container.

Corresponding reference characters indicate corresponding partsthroughout the several views.

DETAILED DESCRIPTION OF THE DRAWINGS

The embodiments disclosed below are not intended to be exhaustive or tolimit the invention to the precise forms disclosed in the followingdetailed description. Rather, the embodiments are chosen and describedso that others skilled in the art may utilize their teachings. While thepresent disclosure is primarily directed to the transfer of crop tissueexplants within a sterile environment, it should be understood that thefeatures disclosed herein may have application to the transfer of othertypes of tissue or objects.

The term ‘plant tissue culture explants’ also be referred to as “planttissue explants’ or “plant tissue cultures’ refer to any plant tissuegrowing or being maintained on a medium which consists of cells inprotoplasmic continuity. Often these explants are multi-cellularconsisting of morphologically complex structures. Plant tissue cultureexplants can include single cells or protoplasts, cell clusters, calluspieces, embryo-like structures, embryos, ovules, ovaries, anthers,microspores, pollen grains, hypocotyls, cotyledons, leaf segments, stempieces, roots and seeds. Exemplary plant tissue culture explants includemaize and cotton embryos, canola hypocotyls, cotton ovules, soybeancotyledons, tobacco leaf segments and rice callus pieces.

Referring to FIG. 1, a handheld tool 100 is shown. Handheld tool 100 isused in connection with a source of negative pressure 102 in an asepticenvironment 104. In the illustrated embodiment, the aseptic environment104 is provided in a laminar flow hood 106 having a work surface 108. Anexemplary laminar flow hood is the EDGEGUARD® brand laminar flow hoodavailable from The Baker Company located in Sandford, Me. The source ofnegative pressure 102 is provided to aseptic environment 104 through avalve 110 provided in a sidewall 112 of laminar flow hood 106. Anexemplary source of negative pressure 102 is a facility vacuum system.In one embodiment, source of negative pressure 102 has a draw of about27 inches of mercury below atmospheric pressure.

Referring to FIG. 2, handheld tool 100 includes a first flexible fluidconduit 120 having a first end 122 and a second end 124. An interior 126of first flexible fluid conduit 120 is in fluid communication with bothfirst end 122 and second end 124. Returning to FIG. 1, first end 122 iscoupled to a hose barb 128 of valve 110. In one embodiment, firstflexible fluid conduit 120 is made of a polymeric material. In oneembodiment, first flexible fluid conduit 120 is made from anautoclavable material. An exemplary first flexible fluid conduit 120 is180 PVC non-toxic autoclavable Lab/FDA/USP VI Grace (¼″ID) tubingavailable from NALGENE Labware having an office at 75 Panorama CreekDrive, Rochester, N.Y. 14625.

Returning to FIG. 2, handheld tool 100 further includes a filter unit130. Filter unit 130 includes a housing 132 having a filter (not shown)positioned therein and a first hose barb 134 and a second hose barb 136.An interior 138 of filter unit 130 permits fluid to flows throughhousing 132 from first hose barb 134 to second hose barb 136 or inreverse. Second hose barb 136 is coupled to second end 124 of firstflexible fluid conduit 120. An exemplary filter unit 130 is Millipore 50mm Millex Sterile Filter Unit stepped hose barb with female Luer slipinterior available from Millipore having an office at 290 Concord Road,Billerica, Mass. 01821.

Handheld tool 100 further includes a second flexible fluid conduit 150having a first end 152 and a second end 154. An interior 156 of secondflexible fluid conduit 150 is in fluid communication with both first end152 and second end 154. Returning to FIG. 1, first end 152 is coupled tohose barb 134 of filter unit 130. In one embodiment, second flexiblefluid conduit 150 is made of a polymeric material. In one embodiment,first flexible fluid conduit 120 is made from an autoclavable material.An exemplary second flexible fluid conduit 150 is the 180 PVC non-toxicautoclavable Lab/FDA/USP VI Grace (¼″ID) tubing available from NALGENELabware.

Handheld tool 100 further includes a coupler 160 having a first end 162and a second end 164. An interior 166 of coupler 160 is in fluidcommunication with both first end 162 and second end 164. Second end 164includes a hose barb which is coupled to second end 154 of secondflexible fluid conduit 150 as shown in FIG. 1. An exemplary coupler isthe Fisher brand Polyethylene Quick Disconnects® 15-315-27D availablefrom Fisher Scientific located at 2000 Park Lane Drive in Pittsburgh,Pa. 15275.

Referring to FIG. 3, handheld tool 100 further includes a tip member 170having a first end 172 and a second end 174. An interior 176 of tipmember 170 is in fluid communication with first end 172 and second end174. Tip member 170 is shown having a generally tapered exterior fromfirst end 172 down to second end 174. Other shapes of tip member 170 maybe used. in one embodiment, tip member 170 is a standard pipette tip. Anexemplary pipette tip is the LTS 1000 ul wide-bore pipette tip availablefrom Rainin Instrument, LLC, located at 7500 Edgewater Drive, P.O. Box2160 in Oakland, Calif.

Referring to FIG. 4, interior of tip member 170 includes a first region180 sized to press-fit with first end 162 of coupler 160. Tip member 170may be coupled to coupler 160 in other manners as well. Suitable methodsfor coupling include mating threads and other suitable methods. In oneembodiment, interior 176 includes a filter 182 positioned in a region184.

As shown in FIG. 4, interior of tip member 170 terminates in an opening186 at second end 174 of tip member 170. Opening 186 is sized to preventthe entrance of a first tissue explant 190A into interior of tip member170. As explained herein, source of negative pressure 102 draws air ininterior 176 generally in direction 192 which results in first tissueexplant 190A becoming coupled to second end 174 of tip member 170. Basedon the amount of draw of source of negative pressure 102, opening 186 issized to provide a sufficient surface area to couple first tissueexplant 190A to second end 174 of tip member 170. In one embodiment,source of negative pressure 102 draws about 27 inches of mercury belowatmospheric pressure and opening 186 is about 40 thousandths of an inchin diameter. Of course different amounts of draw of source of negativepressure 102 and sizes of opening 186 may be used as long as they aresufficient to couple first tissue explant 190A to tip member 170.

As mentioned above, tip member 170 also includes vent passage. As shownin FIG. 4, when vent passage is open to atmosphere air is drawn bysource of negative pressure 102 through both vent passage 178 andopening 186. Vent passage 178 is sized such that even if first tissueexplant 190A is contacting second end 174 of tip member 170 a sufficientamount of air is provided through vent passage 178 to prevent thecoupling of first tissue explant 190A to tip member 170. In oneembodiment, vent passage 178 is about 0.08 inches in diameter (area ofabout 0.02 square inches) while opening 186 is about 0.04 inches indiameter (area of about 0.005 square inches). In one embodiment, thearea of vent passage 178 is about four times the area of opening 186. Inone embodiment, the area of vent passage 178 is at least about fourtimes the area of opening 186. In one embodiment, the area of ventpassage 178 is greater than the area of opening 186.

Referring to FIG. 5, handheld tool 100 is being held in a hand 200 of anoperator 202. First end 172 of tip member 170 and coupler 160 (notshown) are being held between a thumb 204 and middle finger (not shown)of operator 202. An index finger 206 of operator 202 is positioned abovetip member 170. As explained herein, index finger 206 is positioned suchthat index finger 206 may cover vent passage 178 during the use ofhandheld tool 100.

Also shown in FIG. 5 is a first container 194 including a plurality ofplant tissue explants 190, four plant tissue explants 190A-D beingillustrated. The plurality of plant tissue explants 190 are supported ona nutrient medium 196. Exemplary nutrient mediums include a mixture ofinorganic salts, vitamins, a carbon source such as sucrose, and plantgrowth regulators. Plurality of plant tissue explants 190 are spacedapart in first container 194. Generally first container 194 includes alid which is placed over a dish of first container 194 when plurality ofplant tissue explants 190 are not being transferred. A second container198 is also shown in FIG. 5 having a nutrient medium 199. The pluralityof plant tissue explants 190 are being transferred from first container194 to second container 198 to provide a fresh nutrient medium 199 forthe plurality of plant tissue explants 190.

The use of handheld tool 100 will now be explained with references toFIGS. 4-9. An operator 202 positions first container 194 and secondcontainer 198 on work surface 108 of laminar flow hood 106. As such,first container 194 and second container 198 are positioned in anaseptic environment. The operator 202 then grasps handheld tool 100 asshown in FIG. 5 and prepares to transfer plurality of plant tissueexplants 190, one at a time, from first container 194 to secondcontainer 198. In one embodiment, handheld tool 100 includes multipletip members so that multiple tissue explants 190 may be coupled at thesame time.

It should be noted that if a prior transfer of plant tissue material wasjust completed with handheld tool 100, a new or cleaned tip member 170would be first assembled to coupler 160 and the prior tip member 170would be cleaned or discarded. In one embodiment, tip member 170 is madeof an autoclavable material and is cleaned in an autoclave devicesubsequent to use. The use of a new or cleaned tip member 170 maintainsthe aseptic environment between transfer runs.

Referring to FIG. 5, a stop device 210 blocks vent passage 178 fromcommunicating with the atmosphere. As shown in FIG. 4, in the absence ofthe presence of stop device 210 blocking vent passage 178, air flowsinto tip member 170 through both vent passage 178 and opening 186 due tothe draw of source of negative pressure 102. When stop device 210 blocksvent passage 178 the air flow into tip member 170 through vent passage178 is greatly reduced or prevented. This results in the air mainlyentering tip member 170 through opening 186. As second end 174 of tipmember 170 is brought closer to first tissue explant 190A, first tissueexplant 190A is coupled to opening 186 due to the draw of source ofnegative pressure 102, as shown in FIGS. 6 and 7. First tissue explant190A now moves with tip member 170 such that it may be separated fromnutrient medium 196 and moved to second container 198 (see FIG. 9) andplaced upon nutrient medium 199. Once placed on nutrient medium 199,first tissue explant 190A is uncoupled from tip member 170 moving stopdevice 210 to allow more air in through vent passage 178. This reducesthe draw experienced by first tissue explant 190A at opening 186 andcauses first tissue explant 190A to be released. This process isrepeated for each of the plurality of plant tissue explants 190 in firstcontainer 194. In one embodiment, operator 202 transfers all ofplurality of plant tissue explants 190 from first container 194 tosecond container 198. In one embodiment, operator 202 transfers only aportion of plurality of plant tissue explants 190 from first container194 to second container 198 and discards the remainder. In oneembodiment, in the early transfer runs (first few items the plurality ofplant tissue explants 190 are transferred to a new container) all ofplurality of plant tissue explants 190 are transferred to the nextcontainer. As time goes on, the operator 202 based on their professionaljudgment may select less than all of the plurality of plant tissueexplants 190 for transfer the next time around.

As shown in FIG. 7, stop device 210 is illustratively index finger 206of hand 200. (Of course, operator 202 may be wearing gloves or attirewhich would contact tip member 170.) Thus, an operator 202 may couplefirst tissue explant 190A by simply lowering index finger 206 to covervent passage 178 and release first tissue explant 190A by simply raisingindex finger 206 to be spaced apart from vent passage 178. Thisrepetitive motion is less fatiguing on operator 202 over the course ofthe day than the use of forceps as described herein. Other types of stopdevice 210 may be used. In one embodiment, operator 202 may actuate avalve to block and unblock vent passage 178. In one embodiment, operator202 may cover vent passage 178 with a lid or other type of cover toblock vent passage 178 and remove the same to unblock vent passage 178.

As stated above, tip member 170 may be uncoupled from the remainder ofhandheld tool 100 once a transfer of plurality of plant tissue explants190 from first container 194 to second container 198 is complete. Tipmember 170 is then cleaned or discarded. Another tip member 170 iscoupled to the remainder of handheld tool 100 and a subsequent transferrun of other plurality of plant tissue explants 190 is performed.

Referring to FIG. 10, another handheld tool 250 is shown. Handheld tool250 is identical to handheld tool 100 from coupler 160 back to firstflexible fluid conduit 120. Handheld tool 250 also includes a tip member252 which is identical to tip member 170, except that vent passage 178is not included. Tip member 252 includes a first end 254 and a secondend 256 in fluid communication with the first end 254 through aninterior 257 of tip member 252. Tip member 252 includes an opening 258which operates the same as opening 186.

In one embodiment, tip member 252 is a pipette tip. An exemplary pipettetip is the LTS 1000 ul wide-bore pipette tip available from RaininInstrument, LLC, located at 7500 Edgewater Drive, P.O. Box 2160 inOakland, Calif. In the case of tip member 170, in one embodiment, thepipette tip is altered to include vent passage. In contrast, tip member252 may be an off-the-shelf pipette tip.

Interposed between coupler 160 and tip member 252 is a pen-shapedinstrument 262 having a first end 264 and a second end 266. An interior268 of pen-shaped instrument 262 is in fluid communication with bothfirst end 264 and second end 266. Pen-shaped instrument 262 includes avent passage 260. Pen-shaped instrument 262 is shown being generally atapered cylinder. However, pen-shaped instrument 262 may have anysuitable shape for comfortable holding by the hand 200 of the operator202.

Referring to FIGS. 11 and 12, the operation of handheld tool 250 isexplained. An operator 202 positions first container 194 and secondcontainer 198 on work surface 108 of laminar flow hood 106. As such,first container 194 and second container 198 are positioned in anaseptic environment. The operator 202 then grasps handheld tool 250 asshown in FIG. 12 and prepares to transfer plurality of plant tissueexplants 190, one at a time, from first container 194 to secondcontainer 198. It should be noted that if a prior transfer of planttissue material was just completed with handheld tool 250, a new orcleaned tip member 252 would be first assembled to pen-shaped instrument262 and the prior tip member 252 would be cleaned or discarded. In oneembodiment, tip member 252 is made of an autoclavable material and iscleaned in an autoclave device subsequent to use. The use of a new orcleaned tip member 252 maintains the aseptic environment betweentransfer runs.

As shown in FIG. 12, a plurality of tip members 252 are stored in astorage container 280. Storage container 280 includes a plurality ofrecesses 282 into which second end 256 of tip member 252 are placed forstorage. Since a different tip member 252 is used for each transfer runand the remainder of handheld tool 250 does not contact the plurality ofplant tissue explants 190 of a prior transfer run, an operator 202 maycomplete several successive transfer runs without the need for cleaninghandheld tool 250 between runs while still maintaining an asepticenvironment for each run.

To transfer a first tissue explant 190A from first container 194 tosecond container 198, operator 202 positions second end 256 adjacent tofirst tissue explant 190A and blocks vent passage 260 of pen-shapedinstrument 262 with a stop device 210. As explained herein, in oneembodiment, stop device 210 may be index finger 206 of operator 202.Once vent passage 260 is blocked first tissue explant 190A is coupled tosecond end 256 due to the draw of source of negative pressure 102. Firsttissue explant 190A now moves with tip member 252 such that it may beseparated from nutrient medium 196 and moved to second container 198 andplaced upon nutrient medium 199. Once placed on nutrient medium 199,first tissue explant 190A is uncoupled from tip member 252 by movingstop device 210 to allow more air in through vent passage 260 inpen-shaped instrument 262. This reduces the draw experienced by firsttissue explant 190A at opening 258 and causes first tissue explant 190Ato be released. This process is repeated for each of the plurality ofplant tissue explants 190 in first container 194. In one embodiment,operator 202 transfers all of plurality of plant tissue explants 190from first container 194 to second container 198. In one embodiment,operator 202 transfers only a portion of plurality of plant tissueexplants 190 from first container 194 to second container 198 anddiscards the remainder. In one embodiment, in the early transfer runs(first few instances that the plurality of plant tissue explants 190 aretransferred to a new container) all of plurality of plant tissueexplants 190 are transferred to the next container. As time goes on, inlater transfer runs the operator 202 based on their professionaljudgment may select less than all of the plurality of plant tissueexplants 190 for transfer the next time around.

Referring to FIG. 13, a system 300 is illustrated for automaticallytransferring plant tissue explants from a first container 194 to asecond container 198. System 300 performs a transfer run for a pluralityof containers in a serial timeframe. In one embodiment, system 300 ishoused in a housing which provides an aseptic environment fortransferring plant tissue explants.

System 300 includes a first presentment system 302, a second presentmentsystem 304, a robot system 306, and an identification system 308. Firstpresentment system 302 selects a first container 194 from a first queueof containers 310 and moves first container 194 to a region 312. In oneembodiment, presentment system 302 also prepares first container 194 sothat plurality of plant tissue explants 190 may be accessed by robotsystem 306. For example, first container 194 may include a dish 314supporting nutrient medium 196 and plurality of plant tissue explants190 and a lid 316 covering dish 314. First presentment system 302, inthis example, separates lid 316 from dish 314 so that robot system 306may access plurality of plant tissue explants 190. In one embodiment,first presentment system 302 lowers dish 314 relative to lid 316 to gainaccess to the plurality of plant tissue explants 190.

In one embodiment, first presentment system 302 includes a turntable 324which is moved by a motor 314. Turntable 324 of first presentment system302 supports first container 194 which is removed from queue ofcontainers 310 and moves it to region 312 wherein robot system 306interacts with plurality of plant tissue explants 190 and then turntable324 moves it to a second queue 326 of containers. An exemplary systemfor selecting a container from a vertical queue of containers, movingthat container to a region for processing (filling with agar), and thenmoving the container to a second vertical queue of containers isdisclosed in U.S. Pat. No. 4,170,861, the disclosure of which isexpressly incorporated by reference herein. Another exemplary system isMEDIAJET petri dish filler available from INTEGRA Biosciences AG ofSchönbühlstr. 8, CH-7000 Chur Switzerland. First presentment system 302,in one embodiment, also supports additional queues 328 of containerswhich may be processed once queue of containers 310 is completed. Asshown in FIG. 13, turntable 324 includes a plurality of holders A-D forinteracting with a container. As illustrated, holder A removes acontainer from queue 310. Holder B is for presenting an opened containerto robot system 306. Holder C is for placing the container in queue 326.Holder D removes a container from queue 328 once queue 310 is exhausted.

Second presentment system 304 is generally identical to firstpresentment system 302. Second presentment system 304 selects a secondcontainer 198 from a queue of containers 340 and moves second container198 to region 312. In one embodiment, presentment system 304 alsoprepares second container 198 so that plurality of plant tissue explants190 may be placed within second container 198 by robot system 306. Forexample, second container 198 may include a dish 314 supporting nutrientmedium 199 and plurality of plant tissue explants 190 (once transferredby robot system 306) and a lid 316 covering dish 314. Second presentmentsystem 304, in this example, separates lid 316 from dish 314 so thatrobot system 306 may place plurality of plant tissue explants 190 withinsecond container 198. In one embodiment, second presentment system 304lowers dish 314 relative to lid 316.

In one embodiment, second presentment system 304 includes a turntable350 which is moved by a motor 352. Turntable 350 of second presentmentsystem 304 supports second container 198 received from queue ofcontainers 340 and moves it to region 312 wherein robot system 306places plurality of plant tissue explants 190 within second container198 and then moves it to a second queue 356 of containers. Thecontainers in second queue 356 are held until removed by an operator forreturn to the environment designed for the maturing of plurality ofplant tissue explants 190. As mentioned above, an exemplary system forselecting a container from a vertical queue of containers, moving thatcontainer to a region for processing (filling with agar), and thenmoving the container to a second vertical queue of containers isdisclosed in U.S. Pat. No. 4,170,861, the disclosure of which isexpressly incorporated by reference herein. Second presentment system304, in one embodiment, also supports additional queues 328 ofcontainers which may be processed once queue of containers 310 iscompleted. As shown in FIG. 13, turntable 350 includes a plurality ofholders E-H for interacting with a container. As illustrated, holder Eremoves a container from queue 340. Holder F is for presenting an openedcontainer to robot system 306. Holder G is for placing the container inqueue 356. Holder H removes a container from an additional queue oncequeue 340 is exhausted.

Robot system 306 may be any sort of robot system capable of moving a tipmember 252 into proximity of plurality of plant tissue explants 190 infirst container 194 and then moving the plurality of plant tissueexplants 190 to second container 198. One exemplary robot systemincludes an articulated arm. Robot system 306 is controlled by acontroller 370 which executes software instructions stored on a computerreadable medium to perform the operations of robot system 306. Robotsystem 306 supports a fluid conduit 372 which is coupled to source ofnegative pressure 102 through a valve 374. Valve 374 is operativelycoupled to controller 370 which opens valve 374 when a respectiveplurality of plant tissue explants 190 is to be coupled to tip member252 and closes valve 374 when the respective plurality of plant tissueexplants 190 is to be separated from tip member 252. In one embodiment,valve 374 couples and uncouples tip member 252 to/from source ofnegative pressure 102. In one embodiment, valve 374 opens and closes avent passage which effectively increases and decreases, respectively,the amount of draw source of negative pressure 102 has at tip member252.

Robot system 306 includes at least one camera 380 which monitors region312. Based on the images captured by at least one camera 380, controller370 is able to guide the movement of tip member 252. In one embodiment,plurality of plant tissue explants 190 are arranged in a predefinedarrangement 384 (illustratively two rows of three in FIG. 13) in firstcontainer 194 when presented in region 312. Controller 370 moves tipmember 252 so that the plurality of plant tissue explants 190 maintainthis same predefined arrangement 384 in second container 198). Once allof the plurality of plant tissue explants 190 have been transferred fromfirst container 194 to second container 198, controller 370 moves robotsystem 306 to place tip member 252 in a waste receptacle or to becleaned receptacle and couples a sterile tip member 252 to fluid conduit372 from a queue of tip member 252, represented by 388.

Controller 370 is further operatively coupled to identification system308. Identification system 308 includes a reader 390 which identifiesfirst container 194 to controller 370. In one embodiment, firstcontainer 194 includes identifying indicia on dish 314 or lid 316 offirst container 194 and reader 390 reads the indicia. In one example,the indicia includes a bar code and reader 390 reads the bar code andprovides this information to controller 370.

Identification system 308 also includes a marker unit 394 which placesindicia 393 on at least on one of dish 314 and lid 316 of secondcontainer 198. In one embodiment, controller 370 instructs marker unit394 as to what indicia to place on second container 198. In one example,the indicia includes a bar code. An exemplary system for placingidentifying information on second container 198 is disclosed in U.S.Pat. No. 4,572,067, the disclosure of which is expressly incorporated byreference herein.

Referring to FIG. 14, a method 400 of transferring plant tissue explantsis provided. In one embodiment, method 400 is performed with system 300.A region is monitored with at least one camera, as represented by block402. A first container with a plurality of plant tissue explantssupported on a first nutrient substrate for transfer is provided in themonitored region, as represented by block 404. In one embodiment, thefirst container is automatically provided in the monitored region with apresentment system.

In one embodiment, the presentment system includes a robot arm whichretrieves the first container and places it in the monitored region.Referring to FIG. 15, in one embodiment, the presentment system performsthe method 420 wherein the presentment system identifies the firstcontainer, as represented by block 422. In one embodiment, thepresentment system includes a reader which provides input to acontroller of markings or other indicia associated with first container194. The presentment system also selects first container 194 from aqueue of containers, as represented by block 424. The presentment systemfurther removes a lid of first container 194, as represented by block426, and moves first container 194 to the monitored region, asrepresented by block 428.

Returning to FIG. 14, a second container is provided in the monitoredregion for receiving the plurality of plant tissue explants andsupporting the plurality of plant tissue explants on a second nutrientsubstrate, as represented by block 406. In one embodiment, the secondcontainer is automatically provided in the monitored region with apresentment system.

In one embodiment, the presentment system includes a robot arm whichretrieves the second container and places it in the monitored region. Inone embodiment, the presentment system includes a robot arm whichretrieves the second container and places it in the monitored region.Referring to FIG. 16, in one embodiment, the presentment system performsthe method 430 wherein the presentment system selects second container198 from a queue of containers, as represented by block 432, andidentifies the second container 198, as represented by block 434. In oneembodiment, the presentment system includes a marker which based oninput from a controller provides markings or other indicia on secondcontainer 198. The presentment system further removes a lid of secondcontainer 198, as represented by block 436, and moves second container198 to the monitored region, as represented by block 438.

Returning to FIG. 14, the plant tissue explants are automaticallytransferred from the first container to the second container throughnegative pressure applied by a robot system, as represented by block408. Once the plurality of plant tissue explants 190 have beentransferred first container 194 may be discarded.

In one embodiment, the robot system includes a fluid conduit in fluidcommunication with a source of negative pressure. Referring to FIG. 17,in one embodiment the automatic transfer of the plant tissue explantsfrom the first container to the second container through negativepressure applied by a robot system is performed by method 450. The robotsystem selects a tip member from a queue of tip members, as representedby block 452. The robot system is coupled the tip member such that afirst end of the tip member is in fluid communication with the fluidconduit, as represented by block 454. Once the tip member is in place,the robot system transfers the plant tissue explants from firstcontainer 194 to second container 198. For each plant tissue explant inthe first container, the robot system locates the respective planttissue explant in the first container, as represented by block 456 andpositions the first end of the tip member proximate the respective planttissue explant, as represented by block 458. Further, the robot systemcouples the respective plant tissue explant to the first end of the tipmember, as represented by block 460. The respective plant tissue explantis coupled due to the first end of the tip member being in fluidcommunication with the source of negative pressure through a fluidconduit of the tip member. The robot system then moves the tip memberand the respective plant tissue explant to a predefined locationrelative to the second container, as represented by block 462 anduncouples the respective plant tissue explant from the tip member bychanging the pressure in the fluid conduit of the tip member, asrepresented by block 464. In one embodiment, the pressure in the fluidconduit of the tip member is changed by blocking access to or lesseningthe draw of source of negative pressure 102. In one embodiment, thepressure in the fluid conduit of the tip member is changed by unblockinga vent passage in the fluid conduit of the robot system. Once thetransfer from first container 194 to second container 198 is completefor all of the plurality of plant tissue explants 190 in first container194 then the tip member is uncoupled from the fluid conduit of the robotsystem and discarded, as represented by block 468.

While this invention has been described as relative to exemplarydesigns, the present invention may be further modified within the spiritand scope of this disclosure. Further, this application is intended tocover such departures from the present disclosure as come within knownor customary practice in the art to which this invention pertains.

1. A method of transferring plant tissue explants from a first containerhaving a first nutrient medium substrate adapted to provide nutrients tothe plant tissue explants to a second container having a second nutrientmedium substrate adapted to provide nutrients to the plant tissueexplants, the method comprising the steps of: (a) coupling a first planttissue explant to a handheld tool, the handheld tool coupling the firstplant tissue explant through a negative pressure present in an interiorof the handheld tool; (b) generally separating the first plant tissueexplant from the first nutrient medium substrate adapted to providenutrients to the first plant tissue explant and from the firstcontainer; (c) locating the first plant tissue explant relative to thesecond nutrient medium substrate adapted to provide nutrients to thefirst plant tissue explant of the second container; and (d) uncouplingthe first plant tissue explant from the handheld tool.
 2. The method ofclaim 1, wherein the handheld tool includes a fluid conduit having afirst end and a second end, the second end of the fluid conduit being influid communication with the first end of the fluid conduit and thefluid conduit having at least one vent passage positioned between thefirst end of the fluid conduit and the second end of the fluid conduitand being in fluid communication with the first end of the fluid conduitand the second end of the fluid conduit and to the surroundingenvironment, the vent passage being blocked from fluid communicationwith the surrounding environment during steps (a) through (c) with astop device and the vent passage being in fluid communication with thesurrounding environment during step (d).
 3. The method of claim 2,wherein stop device is one of an operator's finger and a devicesupported by the operator's finger.
 4. The method of claim 2, whereinthe fluid conduit is an assembly and includes at least a first portionand a second portion, both the first portion and the second portionhaving a respective interior in fluid communication with the first endof the handheld tool and the second end of the handheld tool when thefirst portion and the second portion are assembled, the method furthercomprising the steps of: (e) providing a plurality of first portions ofthe handheld tool; (f) assembling a first one of the plurality of firstportions of the handheld tool to the second portion of the handheldtool; (g) performing steps (a) through (d) for all of the plant tissueexplants in the first container selected for transfer to the secondcontainer; (h) separating the first one of the plurality of firstportions of the handheld tool from the second portion of the handheldtool; (i) obtaining a third container including another plurality ofplant tissue explants for transfer to a fourth container; (j) assemblinga second one of the plurality of first portions of the handheld tool tothe second portion of the handheld tool; and (k) performing steps (a)through (d) for all of the plant tissue explants in the third containerselected for transfer to the fourth container.
 5. The method of claim 4,wherein the plurality of first portions are a plurality of pipette tips.6. The method of claim 5, wherein the plurality of pipette tips arestored generally vertical in a pipette tip box with a tip portion lowerthan a coupling portion, the step of assembling a first one of theplurality of first portions of the handheld tool to the second portionof the handheld tool includes the step inserting a portion of the secondportion of the handheld tool into the coupling portion of a firstpipette tip of the plurality of pipette tips while the first pipette tipis stored in the pipette tip box.
 7. The method of claim 1, whereinsteps (a) through (d) are performed in an aseptic environment.
 8. Themethod of claim 7, wherein steps (a) through (d) are performed in alaminar flow hood.
 9. The method of claim 1, wherein the step ofuncoupling the first plant tissue explant from the handheld toolincludes the step of reducing the negative pressure in the handheld toolby opening a vent passage of the handheld tool.
 10. The method of claim1, further comprising the step of preventing the first plant tissueexplant from entering into an interior of the handheld tool.
 11. Themethod of claim 10, wherein the first plant tissue explant is coupled toa first end of the handheld tool, the first end of the handheld toolincluding an opening sized to prevent the first plant tissue explantfrom entering into the interior of the handheld tool.
 12. The method ofclaim 1, wherein during step (c) the first plant tissue explant is heldstationary relative to the handheld tool.
 13. The method of claim 2,further comprising the step of filtering the fluid as it travels betweenthe first end of the fluid conduit and the second end of the fluidconduit.